Williams S P, Bridger W A
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1987 Jul 14;26(14):4483-7. doi: 10.1021/bi00388a046.
Succinyl-CoA synthetase of Escherichia coli has an alpha 2 beta 2 subunit structure. The enzyme shows strict half-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha subunit that represents a step in catalysis. Several lines of evidence indicate that this behavior may result from cooperative interactions between alternatingly functional active sites, so that subsequent steps in catalysis at one site may be promoted by phosphoryl transfer to the site on the neighboring half of the molecule. This study is directed toward learning more about the nature of these cooperative interactions. Here we have used positional isotope exchange (i.e., exchange of 18O between the beta, gamma bridge and the beta nonbridge position of ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was prepared in which one of the two active sites was thiophosphorylated; this species thus has one of its two active-site histidine residues occupied and unavailable for further reaction with ATP. Treatment of this monothiophosphorylated enzyme with [beta, gamma-18O]ATP resulted in no significant scrambling of isotope into the nonbridge position, clearly indicating that the enzyme does not undergo even transient bisphosphorylation. We interpret the results in terms of a model of catalysis in which phosphoryl transfer to the second site occurs in concerted fashion with transfer from the first.
大肠杆菌的琥珀酰辅酶A合成酶具有α₂β₂亚基结构。该酶在α亚基中一个组氨酸残基的磷酸化方面表现出严格的半位点反应性，这是催化过程中的一个步骤。几条证据表明，这种行为可能源于交替发挥功能的活性位点之间的协同相互作用，因此一个位点催化过程中的后续步骤可能会因磷酰基转移到分子相邻半部分的位点而得到促进。本研究旨在更深入了解这些协同相互作用的本质。在这里，我们使用了位置同位素交换（即ATP的β、γ桥与β非桥位置之间的¹⁸O交换）作为瞬时双磷酸化的测试。制备了琥珀酰辅酶A合成酶，其中两个活性位点之一被硫代磷酸化；因此，该物种的两个活性位点组氨酸残基之一被占据，无法与ATP进一步反应。用[β,γ-¹⁸O]ATP处理这种单硫代磷酸化酶，没有导致同位素明显混入非桥位置，这清楚地表明该酶甚至不会经历瞬时双磷酸化。我们根据一种催化模型来解释这些结果，在该模型中，磷酰基向第二个位点的转移与从第一个位点的转移协同发生。
